The structure of the complex between the arsenite oxidase from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c 552

The crystal structure of the electron transfer complex between arsenite oxidase (AioAB) from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c 552 (cytc 552) is presented. Cytc 552 docks within a cleft at the interface of the AioA and AioB subunits, which allows a close association between redox cofactors.; this close association presumably facilitates fast electron transfer and underpins the ability of this organism to respire in arsenic contaminated environments.


Introduction
Interprotein electron transfer processes are key biochemical events which play critical roles in fundamental biological processes such as photosynthesis, respiration and nitrogen fixation (Berg et al., 2007). The structural characterization of protein-protein complexes that participate in electron transfer is challenging due to their weak and transient nature (Supplementary Table S1; Antonyuk et al., 2013). The formation of electron transfer complexes requires efficient and finely tuned docking and dynamics at the protein-protein interface (Bendall, 2020;Moser et al., 1992), with complexes being guided together by complementary electrostatic steering and the relative positions of the proteins being tuned through hydrophobic/van der Waals interactions (Leys & Scrutton, 2004). The distance between the redox centers within these complexes has been shown to influence the rate of electron transfer (Davidson, 2000;Marcus & Sutin, 1985;Moser et al., 1992).
Interprotein electron transfer processes are crucial for the survival of arsenic respiring organisms cultured from arsenic contaminated environments (Santini et al., 2007). Arsenic is toxic to most organisms in its inorganic forms arsenite (AsO 3À 3 ) and arsenate (AsO 3À 4 ) (Bissen & Frimmel, 2003;Domingo, 1995); however, prokaryotes such as Pseudorhizobium banfieldiae sp. strain NT-26 can catalyze the aerobic oxidation of arsenite (to arsenate) through the action of the enzyme arsenite oxidase (AioAB). The physiological electron acceptor for the AioAB enzyme has been shown to be a soluble c-type cytochrome (cytc 552 ; Santini et al., 2007;Santini & vanden Hoven, 2004). The AioAB complex consists of two subunits: a large subunit, AioA (93 kDa), which contains a molybdenum cofactor (Moco) at the active site and a [3Fe-4S] cluster, and a small subunit, AioB (14 kDa), which contains a Rieske [2Fe-2S] cluster. AioAB is a member of the dimethyl sulfoxide (DMSO) reductase family of molybdoenzymes. The crystal structure of AioAB from P. banfieldiae sp. strain  has been determined and refined to 2.7 Å resolution (PDB 4aay; Warelow et al., 2013). The proposed reaction mechanism of AioAB comprises oxidation of arsenite at the molybdenum site [reducing molybdenum(VI) to molybdenum(IV)], which releases two electrons that transfer one at a time to the [3Fe-4S] cluster of the AioA subunit and then to the [2Fe-2S] Rieske cluster of the AioB subunit. The electrons are then received by the electron acceptor cytc 552 (Bernhardt & Santini, 2006;Santini & Ward, 2018;Supplementary Fig. S1).
To investigate the structural basis of the electron transfer process that underpins respiration using arsenite, here we report the crystal structure of the AioAB/cytc 552 complex from P. banfieldiae sp. strain NT-26. We show that cytc 552 sits within a cleft at the interface between the AioA and AioB subunits, with a relatively short distance between redox-active cofactors. The crystals show an interesting arrangement, with three of the four cytc 552 molecules located in a 'functional' location. The positioning of the fourth cytc 552 seems to be 'nonfunctional' and presumably facilitates crystal packing.

Protein overexpression and purification
The pPROEX-HTb-AioBA plasmid was transformed into Escherichia coli strain DH5 (New England Biolabs). Cultures were grown aerobically at 21 C in Luria broth (LB) with 1 mM sodium molybdate and supplemented with ampicillin (100 mg ml À1 ) with slow shaking ($50 rev min À1 ). The cultures were induced with 40 mM isopropyl -d-thiogalactopyranoside (IPTG) and were harvested after 24 h.
His-AioBA was purified by immobilized metal-affinity chromatography (IMAC) followed by size-exclusion chromatography (SEC). Frozen cell pellets were thawed and resus-pended in binding buffer (20 mM potassium phosphate, 500 mM NaCl, 20 mM imidazole pH 7.3). The cells were lysed using a TS series bench-top cell disruptor (Constant Systems) at 241 MPa and insoluble debris was removed by centrifugation (Beckman JLA-25.50; 30 000g, 1 h, 4 C). The soluble fraction was incubated with 5 ml Ni-Sepharose 6 Fast Flow resin (Cytiva; 4 C; 1 h stirring) that had been pre-equilibrated with binding buffer (20 mM potassium phosphate, 500 mM NaCl, 20 mM imidazole pH 7.3). The resin was washed with ten column volumes (CV) of binding buffer, followed by elution of bound protein with elution buffer (20 mM potassium phosphate, 500 mM NaCl, 500 mM imidazole pH 7.3; 5 CV). The eluent was dialyzed (3000 molecular-weight cutoff SnakeSkin Dialysis Tubing, ThermoScientific) against 2.0 l dialysis buffer (50 mM MES pH 5.5) overnight. This resulted in the precipitation of contaminating proteins, which were removed by centrifugation (30 000g, 30 min). The supernatant was concentrated by centrifugal ultrafiltration (10 000 molecular weight cutoff, Millipore Amicon Ultra) and further purified by SEC (HiLoad 16/600 Superdex 200 pg, Cytiva; 4 C) in 50 mM MES, 100 mM NaCl pH 5.5. The purest fractions as determined by SDS-PAGE were pooled and concentrated to approximately 10 mg ml À1 by centrifugal ultrafiltration. Aliquots of the purified protein (which will be referred to as AioAB in the following) were snap-frozen and stored at À80 C until further use. The concentration of the AioAB enzyme was measured spectrophotometrically at 682 nm with " 682 = 5.6 mM À1 cm À1 (Watson et al., 2017).
The His-cytc 552 protein was purified by cation-exchange chromatography, IMAC and SEC. Frozen cell pellets were thawed at room temperature and resuspended in cell-lysis buffer (20 mM MES pH 5.5). The cells were disrupted by passage through a TS series bench-top cell disruptor (Constant Systems) at 241 MPa. Cell debris was removed by centrifugation (Beckman JLA-25.50; 30 000g, 1 h, 4 C) and the soluble fraction was loaded onto a 5 ml HiTrap SP Sepharose Fast Flow column (Cytiva; 4 C), washed with buffer consisting of 70 mM NaCl, 20 mM MES pH 5.5 and eluted with a linear NaCl gradient (0.07-0.45 M in 50 mM MES pH 5.5). The eluent was dialyzed (3000 molecular weight cutoff SnakeSkin Dialysis Tubing, ThermoScientific) against 2.0 l dialysis buffer (50 mM potassium phosphate, 0.5 M NaCl pH 7.4) overnight. The protein was loaded onto a 5 ml HisTrap column (Cytiva) and eluted using an imidazole gradient (0-0.5 M in 50 mM potassium phosphate, 0.5 M NaCl pH 7.4) followed by SEC (HiLoad 16/600 Superdex 75 pg, Cytiva; 4 C; 20 mM Tris, 150 mM NaCl pH 7.8). The purified protein (which will be referred to as cytc 552 ) was concentrated to 10 mg ml À1 and stored at À80 C until further use. The concentration of research papers oxidized cytc 552 was determined spectrophotometrically at 550 nm using " 550 = 8.7 mM À1 cm À1 (Santini et al., 2007).

Enzyme kinetics
AioAB activity assays were carried out as described previously (Watson et al., 2017). The reduced-oxidized extinction coefficient for cytochrome c 552 at 550 nm is 23 mM À1 cm À1 (Santini et al., 2007) and that at 416 nm is 59 mM À1 cm À 1 (Santini et al., 2007). Purified AioAB enzyme (2 nM) was incubated with fully oxidized cytochrome c 552 (20 mM) in 50 mM Tris-HCl pH 8.0 (Watson et al., 2017) with increasing concentrations of arsenite (0-1 mM). The reaction was followed at 550 nm. The steady-state kinetics with cytc 552 as the substrate were determined using an excess of arsenite (2.5 mM) pre-incubated with purified AioAB enzyme (2 nM), followed by the addition of various concentrations of cytc 552 (0-10 mM). In this case, the reaction was followed at 416 nm (Watson et al., 2017). Kinetic experiments were performed using triplicate measurements and data fitting was carried out using the Michaelis-Menten function with GraphPad Prism version 7.0 for Mac OS X (GraphPad Software, La Jolla, California, USA).

Protein crystallization and data collection
Purified AioAB (in 50 mM Tris, 100 mM NaCl pH 7.8) and cytc 552 (in 50 mM Tris, 100 mM NaCl pH 7.8) were mixed and incubated on ice at a molar ratio of 1:1.5 AioAB:cytc 552 (total protein concentration 5 mg ml À1 ) before crystallization via sitting-drop vapor diffusion in 96-well plates (Molecular Dimensions). The stoichiometry of the mixture (AioAB: cytc 552 ) was based on our previous experience in crystallizing the SorT/SorU complex from Sinorhizobium meliloti (McGrath et al., 2015). Initial crystallization trials for the AioAB/cytc 552 complex were conducted using the Index HT (Hampton Research) and ProPlex (Molecular Dimensions) screens. Drops consisting of equal volumes (0.2 ml) of reservoir solution and protein solution were dispensed by a Crystal Gryphon liquid-handling system (Art Robbins Instruments) and were equilibrated against a 50 ml reservoir of screen solution at 20 C. Multiple plate-like crystals of AioAB/cytc 552 were observed within one week in conditions A4 (0.1 M bis-Tris pH 6.5, 2.0 M ammonium sulfate), E7 [0.05 M magnesium chloride hexahydrate, 0.1 M HEPES pH 7.5, Optimization of these conditions was carried out by hanging-drop vapor diffusion in 24-well VDX plates (Hampton Research), varying the concentrations of sodium chloride (0.1-0.2 M), HEPES (0.05-0.1 M) and PEG 3350 (15-25%) and the pH (6.5-7.5). Diffraction-quality crystals of AioAB/cytc 552 grew after two weeks in drops consisting of equal volumes (2 ml; 1:1) of the AioAB/cytc 552 preparation and reservoir solution [0.2 M sodium chloride, 0.1 M HEPES pH 7.3, 18%(w/v) PEG 3350] equilibrated against 500 ml reservoir solution at 20 C. Crystals were cryoprotected in reservoir solution containing 25%(w/v) glycerol before flashcooling in liquid nitrogen. Crystallization conditions are given in Table 1.

Data collection, structure solution and refinement
Diffraction data were collected from the AioAB/cytc 552 crystals using an EIGER 16M detector on beamline MX2 at 13 000 eV at the Australian Synchrotron. All data were collected at 100 K, processed with XDS (Kabsch, 2010) and merged and scaled with AIMLESS (Evans & Murshudov, 2013). Unit-cell parameters and data-collection statistics are presented in Table 2.
The crystal structure of the AioAB/cytc 552 complex was solved by molecular replacement with MOLREP (Vagin & Teplyakov, 2010)    the water molecules removed. Initial rounds of refinement of a model with four AioAB complexes per asymmetric unit yielded a difference Fourier electron-density map which showed positive difference density for the location of four molecules of cytc 552 per asymmetric unit. These were placed by phased molecular replacement with MOLREP using a search model generated from the structure of ferrocytochrome c 2 (PDB entry 1co6; Badilla et al., 2018) modified by CHAINSAW (Stein, 2008). Manual model building and the addition of water molecules were carried out in Coot (Emsley et al., 2010) with iterative cycles of refinement using REFMAC5 . The geometry of the final model was determined with MolProbity (Chen et al., 2010). Refinement statistics are summarized in Table 3.

Results and discussion
The structure of the AioAB/cytc 552 complex was solved and refined to 2.25 Å resolution (Tables 2 and 3). The structure includes four copies of the AioAB assembly per asymmetric unit, arranged as two A 2 B 2 heterotetramers. The AioA subunits include residues 2-844 and are composed of four domains (domains I, II, III and IV). Domain I is composed of three antiparallel -sheets, domain II and domain III have similar -sandwich topologies and domain IV predominantly consists of six antiparallel -sheets flanked by five small -helices. The AioB subunits include residues 44-175 and have a fold consisting of a six-stranded antiparallel -barrel and a four-stranded antiparallel -sheet. The AioA subunit houses the Moco site, which is a common feature of the DMSO reductase family of molybdenumcontaining enzymes, and the [3Fe-4S] cluster. The Mo atom is coordinated by one oxo ligand and the thiol groups of the two pterin cofactors in an approximate square-pyramidal geometry, with an average Mo O distance across all four copies per asymmetric unit of 1.8 AE 0.1 Å . The [3Fe-4S] cluster is coordinated by a conserved cysteine-rich motif (Cys24-X 2 -Cys27-X 3 -Cys31-X 70 -Ser102) and the AioB subunit houses the [2Fe-2S] Rieske cluster, which is coordinated by two cysteine residues and two histidine residues (Cys103-X-His105-X 15 -Cys121-X 2 -His124).
In addition to the two AioA 2 B 2 complexes, there are four molecules of cytc 552 per asymmetric unit. The cytc 552 protomers are composed of four -helices arranged to form a bundle that frames a heme-binding site. His38 and Met103 are axial ligands of the central Fe atom and the porphyrin ring is covalently attached to Cys34 and Cys37 ( Fig. 2c and Supplementary Fig. S6). This is the first reported crystal structure of cytc 552 from P. banfieldiae sp. strain NT-26. A search of the coordinates of cytc 552 against the Protein Data Bank (PDB) using PDBeFold (Krissinel & Henrick, 2004) reveals similarity to the structures of cytc 552 from Paracoccus denitrificans (PDB entry 1ql4; Harrenga et al., 2000) and cytc 2 from Rhodopseudomonas viridis (PDB entry 1co6; Sogabe & Miki, 1995), with root-mean-square deviation (r.m.s.d.) values of 0.6-0.7 Å (over 92 and 79 common C positions), indicating similar structures.

Two different AioA 2 B 2 /(cytc 552 ) 2 complexes are present in the crystal
In the asymmetric unit, two molecules of cytc 552 are associated with each of the two AioA 2 B 2 assemblies, so there are two AioA 2 B 2 /(cytc 552 ) 2 complexes per asymmetric unit (Fig. 1). In one complex (chains ABI and CDJ) the two cytc 552 molecules (chains I and J) are located at similar relative positions in a cleft near the AioA/AioB interface (Fig. 1a). In the other AioA 2 B 2 /(cytc 552 ) 2 unit (chains EFK and GHL) the relative positions of the two cytc 552 molecules are different. One (chain K) is consistent with that described above, sitting between the AioA and AioB subunits, while the other (chain L) associates with AioA (chain G) from one AioAB heterodimer and AioB (chain F) from the neighboring heterodimer (Fig. 1b).
The three cytc 552 molecules that lie at the AioA/AioB interface (chains I, J and K) are located such that the edge-toedge distance between the [2Fe-2S] Rieske cluster in AioB and the heme in cytc 552 is 7.5 Å , which is consistent with fast electron transfer (discussed further below; Page et al., 1999). The unique cytc 552 (chain L) that associates between heterodimers shows edge-to-edge distances between the cytc 552 heme, the Moco (Mo atom) and the [3Fe-4S] cluster (residue Cys24) of AioA of 25 and 29 Å , respectively. The distance between the cytc 552 heme and the [2Fe-2S] cluster (residue Cys103) in AioB of the neighboring heterodimer is 38 Å (Supplementary Fig. S2). These distances are outside the accepted range for fast electron transfer. The positioning of the unique cytc 552 in the complex therefore does not represent an electron transfer complex. This positioning of cytc 552 presumably facilitates crystallization ( Supplementary Fig. S3  or a functional assembly. This is reminiscent of a previously reported structure of chicken liver sulfite oxidase (Gallus gallus; PDB entry 1sox; Kisker et al., 1997). This enzyme contains three domains: an N-terminal cytochrome domain, a Moco domain and a C-terminal domain. Interestingly, in this structure the cytochrome domain is positioned so that the edge-to-edge distance is 32 Å between the Mo atom and the heme cofactor, which is also outside the range for fast electron transfer. In solution, a flexible linker between the Moco and cytochrome domains allows the cofactors to approach at proximity. The following discussion will therefore describe the AioAB/cytc 552 complex with cytc 552 positioned at the AioA/ AioB interface.
The AioAB/cytc 552 interaction shows significant charge complementarity, with negative charge on the AioAB complex correlating with a concentration of positive charge on the surface of cytc 552 (Fig. 2e). These charged areas encircle neutral surfaces that correlate with the 'footprints' of each electron transfer partner on the other. There are between four and 11 water molecules (over the three AioAB/cytc 552 complexes per asymmetric unit) that sit between AioAB and cytc 552 and which interact with polar and charged surface residues. Superposition of the coordinates of the AioA and AioB subunits from this work with those of AioAB alone (PDB entry 4aay; Warelow et al., 2013), yields r.m.s.d. values of 0.20 and 0.28 Å , respectively (over 832 and 132 common C positions), indicating minimal changes on association with cytc 552 (Supplementary Fig. S4b).
Within the AioAB/cytc 552 complex, the cytc 552 protein shows an average B factor of 44.0 Å 2 , which is significantly higher than that of AioAB (23.6 Å ). In addition, the relative temperature factor per residue for cytc 552 increases with increasing distance from the AioAB/cytc 552 interface ( Supplementary Fig. S4a), indicating that the cytc 552 molecule is dynamic relative to AioAB within the crystalline lattice. This has been observed previously for related electron transfer complexes, such as the SorT/SorU complex from S. meliloti (McGrath et al., 2015). Flexibility in the interactions between protein partners has been proposed to be necessary to achieve optimal orientations for efficient electron transfer (Leys & Scrutton, 2004;van Amsterdam et al., 2002).
Taken together, these observations are consistent with the structures of other comparable electron transfer complexes, which typically feature a modest number of hydrogen bonds and salt bridges at the protein-protein interface and relatively small, buried surface areas between protein partners (Supplementary Table S1). The AioAB/cytc 552 structure is therefore typical of transient complex formation for fast electron transfer (Miyashita et al., 2003).

The electron transfer pathway between AioAB and cytc 552
The vinyl groups on the porphyrin ring of cytc 552 contribute to the AioAB/cytc 552 interface. AioB residues Phe108, Pro109 and Pro122 lie closest to the cytc 552 heme (Fig. 3a). As Two different AioA 2 B 2 /(cytc 552 ) 2 complexes are present in the asymmetric unit of the AioAB/cytc 552 crystal structure. In both of the structures the AioA subunits are shown as gray surfaces, the cytc 552 molecules are in salmon and the AioB subunits are in cyan. (a) The AioA 2 B 2 complex represented by chains ABI and CDJ. The two cytc 552 molecules (chains I and J) are located at similar positions in a cleft near the AioA/AioB interface. (b) The AioA 2 B 2 complex represented by chains EFK and GHL. One molecule of cytc 552 (chain K) is located at the AioA/AioB interface. The second molecule of cytc 552 (chain L) is unique in that it sits between AioAB heterodimers. mentioned previously, the closest edge-to-edge distance between Cys103, which coordinates the Rieske cluster in AioB, and the vinyl group of heme in cytc 552 is 7.5 Å , which is well within the distance for fast electron transfer through the protein medium (Page et al., 1999). HARLEM analysis of the coordinates of the complex (Kurnikov, 2000) predicts that the dominant electron-tunneling pathway from AioB to cytc 552 proceeds from the [2Fe-2S] Rieske cluster in the AioB subunit to Pro122 and across the protein-protein interface to the porphyrin ring and onto the heme iron, with Pro122 at a distance of 4.1 Å from the closest heme vinyl group (Fig. 3a,  Supplementary Table S2). This suggests a role for Pro122 in the electron transfer process, which is consistent with its conservation in the sequences of Rieske cluster-containing proteins and subunits, including arsenite oxidase from Alcaligenes faecalis (PDB entry 1g8k; Ellis et al., 2001), the Rieske protein II SoxF from Sulfolobus acidocaldarius (Sf SoxF; PDB entry 1jm1; Bö nisch et al., 2002), the Rieske protein from Thermus thermophilus (TtRp; PDB entry 1nyk; Hunsicker-Wang et al., 2003) and the Rieske protein involved in photosynthetic and respiratory electron transport in Synechocystis PCC 6803 (SyPetC3; PDB entry 5cxm; Veit et al., 2016;Fig. 3b). The involvement of proline residues in interprotein electron transfer has previously been proposed for the complex between the nitrite reductase from Achromobacter xylos-oxidans GIFU 1051 and its electron acceptor cytochrome c (the AxgNIR/cytc 551 complex; PDB entry 2zon; Nojiri et al., 2009).
Previous studies examining the kinetics of arsenite oxidation catalyzed by AioAB with horse heart cytochrome c (hhcytc) as the electron acceptor revealed that mutation of Phe108 in AioB to Ala led to a 30-fold decrease in the rate of the reaction relative to the native enzyme (Warelow, 2015). These data are consistent with the observation from the present structure that Phe108 lies at the interface between the AioB and cytc 552 proteins (the Phe108-heme distance is 5.2 Å ). Aromatic residues such as phenylalanine have been shown to be involved in electron transfer in other complexes (Hirasawa et al., 1998;Liang et al., 1987), including in the structure of caa 3 -type cytochrome oxidase from T. thermophilus (Lyons et al., 2012). Whether this residue is part of the electron transfer pathway to cytc 552 or facilitates and/or stabilizes complex formation requires further investigation. Notably, this residue is not conserved in the sequences of comparable Rieske proteins (Fig. 3b).

AioAB is catalytically efficient in the presence of cytc 552 in solution
Previous studies have reported the kinetics of arsenite oxidation catalyzed by AioAB using the artificial electron The structure of the AioAB/cytc 552 electron transfer complex. (a) The AioAB/cytc 552 complex as observed for chains ABI, CDJ and EFK (AioA in blue, AioB in cyan and cytc 552 in salmon). (b) The interface between AioAB and cytc 552 . Residues that participate in the two salt bridges are shown. (c) The structure of cytochrome cytc 552 . The four helices are labeled and the heme cofactor is shown in salmon. The heme Fe atom is coordinated by His38 and acceptors DCPIP and hhcytc. To complement our structural analyses, we determined the activity of the AioAB enzyme with its native electron acceptor cytc 552 . In the presence of an excess of cytc 552 , Michaelis-Menten analysis of arsenite oxidation by AioAB monitored spectrophotometrically yielded a K m(arsenite) of 9.06 AE 1.3 mM and a turnover number of 205 AE 19 s À1 . These values are similar to those reported with hhcytc as the electron acceptor (13.0 AE 0.15 mM and 211.2 AE 0.15 s À1 , respectively). Analyses in the presence of an excess concentration of arsenite yielded K m(cytc552) = 2.9 AE 0.2 mM and k cat = 390 AE 25 s À1 (Supplementary Table S3).
The similarity of the turnover numbers for arsenite oxidation with cytc 552 and hhcytc as electron acceptors is interesting given that one protein is a native partner and the other is not. Both proteins show positively charged surfaces and basic pI values (8.7 and 10.0, respectively) and have similar redox potentials [cytc 552 , 275 mV (Kalimuthu et al., 2014); hhcytc, 256-266 mV (Weber et al., 1987)]. Presumably, these features allow hhcytc to substitute for cytc 552 in the in vitro assay. The electrostatic surfaces of other c-type cytochrome electron acceptors which serve as electron transfer partners to members of the molybdenum protein family also have an overall positive charge. The value of K m(cytc552) determined here is similar to those reported for these systems, including chicken liver sulfite oxidase (CSO; G. gallus; Kisker et al., 1997) and the sulfite dehydrogenase SorAB complex from Starkeya novella, with K m(cytc) values between 2 and 4 mM (Kappler et al., 2006;Kappler & Enemark, 2015). A notable exception is the SorT/SorU complex from S. meliloti (McGrath et al., 2015). The electron acceptor SorU has an overall negative charge (pI of $4) and a higher K m(SorU) (32 AE 5 mM).
The k cat values for these systems vary (Supplementary Table  S3, with the AioAB/cytc 552 system apparently being particularly efficient (Brody & Hille, 1999;Kappler et al., 2006). The docking and dissociation of AioAB/cytc 552 before and after electron transfer, respectively, presumably play a significant role in the rate of turnover and can be influenced by the electrostatic complementarity and the number of interactions at the protein-protein interface (Leys & Scrutton, 2004).

Conclusion
The structure of the AioAB/cytc 552 complex reported here shows an interesting combination of 'functional' and 'nonfunctional' assemblies within the crystals. The positioning of the unique cytc 552 molecule between AioAB heterodimers presumably facilitates crystallization but does not represent a fast electron transfer complex. The remaining three AioAB/ cytc 552 modules per asymmetric unit show the cytc 552 molecules positioned in a cleft between the AioA and AioB subunits, with close association between the redox-active cofactors for fast electron transfer.

Related literature
The following references are cited in the supporting information for this article: Axelrod et al.  The electron transfer pathway between AioAB and cytc 552 . (a) Pathway for electron transfer (Kurnikov, 2000). (b) Secondary-structure-based sequence alignment of Rieske proteins. Conserved residues near the Rieske cluster are colored blue and Phe108, which is unique to AioB, is in red. Residue numbers in purple correspond to the AioB sequence. The alignment was generated using Clustal Omega (Sievers et al., 2011). Abbreviations used are as follows: AioB, P. banfieldiae sp. strain NT-26 (this work); AfAioB, AioB subunit of arsenite oxidase from A. faecalis; Sf SoxF, Rieske protein II from S. acidocaldarius; TtRp, Rieske protein from T. thermophilus; SyPetC3, Rieske protein from Synechocystis PCC 6803.